Wound healing compositions and methods using tropoelastin and lysyl oxidase

ABSTRACT

The present invention provides compositions and methods for promoting wound healing. The composition comprises virgin monomers of tropoelastin and lysyl oxidase. When the lysyl oxidase comes in contact with the tropoelastin, cross-linking of the tropoelastin monomers will occur to form elastin. Contacting the tropoelastin and lysyl oxidase together and applying the mixture to a wound before substantial cross-linking has occurred promotes wound healing by holding the damaged tissue together, increasing the rate of healing, and decreasing the amount of scarring.

BACKGROUND OF THE INVENTION

Wound healing is a complex biological process that involves manydifferent cell types, many different cytokines, the extracellular matrix(ECM), and numerous interactions among them. Most wounds heal rapidlyand efficiently within a week or two; however, the result is neitheraesthetically nor functionally perfect. Wound contraction and scarformation are currently unavoidable results of wound healing. Scartissue is less flexible than normal skin and can be cosmeticallydisfiguring, and wound contraction can lead to joint disablement (Lammeet al., J. Histochem. Cytochem. 44:1311, 1996). Scars lack elastin andconsist of poorly reconstituted collagen matrix in dense parallelbundles rather than the mechanically efficient basket-weave meshwork ofcollagen in unwounded dermis (Martin, Science 276:75, 1997). Two majorgoals of wound-healing biology are more rapid wound healing and moreperfect reconstruction of the damaged parts. Compositions and methodsuseful in accomplishing these goals are currently needed.

Wound Healing

Wound healing has been divided into a number of overlapping phases.These include fibrin clot formation, recruitment of inflammatory cells,reepitheliazation, and matrix formation and remodeling. Immediatelyafter tissue injury, blood vessel disruption leads to the extravasationof blood and concomitant platelet aggregation and blood coagulationresulting in fibrin clot formation. Activated platelets trapped withinthe fibrin clot degranulate and release a variety of cytokines andgrowth hormones. These cytokines and growth hormones help to recruitinflammatory cells to the site of injury, to stimulate angiogenesis, andto initiate the tissue movements associated with reepitheliazation andconnective tissue contraction.

Neutrophils and monocytes are recruited to the site of injury by anumber of chemotactic signals including the growth factors and cytokinesreleased by the degranulating platelets, formyl methionyl peptidescleaved from bacterial proteins, and the by-products of proteolysis offibrin and other matrix proteins. Neutrophil infiltration ceases after afew days, but macrophages continue to accumulate by continuedrecruitment of monocytes to the wound site. Activated macrophagesrelease growth factors and cytokines thereby amplifying the earliersignals from the degranulating platelets.

Formation of granulation tissue and reepithelialization of the woundsite begins after several hours (Clark, J. Am. Acad. Dermatol. 13:701,1985). Reepithelialization is performed by the basal keratinocytes whichlose their attachments to the basal lamina and crawl over theprovisional matrix of fibrin and fibronectin, and underlying matrix.Some hours after the onset of migration, epidermal cells begin toreproduce and thereby provide the cells needed to replace those lostduring the injury. Keratinocyte proliferation is regulated bykeratinocyte growth factor and members of the epidermal growth factor(EGF) family. In order to migrate through the fibrin clot, thekeratinocytes must dissolve the fibrin barrier in front of them. Plasminis the chief fibrinolytic enzyme used in this process. Reepitheliazationis made easier by the underlying contractile connective tissue, whichshrinks to bring the wound margins toward one another. Epidermalmigration ceases when the wound surface has been covered by a monolayerof cells.

Cells of the new epidermis undergo the standard differentiation programof cells in the outer layers of unwounded epidermis. A new stratifiedepidermis is, thereby, reestablished from the margins of the woundinward. Matrix formation and remodeling begins simultaneously withreepithelialization. The matrix is constantly altered over the nextseveral months with the elimination of the fibronectin from the matrixand the accumulation of collagen that provides the residual scar withincreasing tensile strength. Elastin fibers, which are responsible forthe elasticity of tissue, are only detected in human scars years afterthe injury (Compton et al., Lab. Invest. 60:600, 1989).

Methods to Promote Wound Healing

In the past, many methods have been proposed and tested to promote woundhealing and limit scarring; however, better methods and compositions arestill needed. These older methods include cyanoacrylate tissueadhesives, a combination of epidermal transplantation and acollagen/elastin dermal substitute, application of collagen andglycosaminoglycans to the site of injury, and biocompatible adhesiveswith collagen.

One of the more popular methods is the use of cyanoacrylate tissueadhesives. These adhesive have been used in place of and in conjunctionwith sutures. Cyanoacrylate adhesives have been used in cases rangingfrom cardiac surgery (Robicsek et al., J. Card. Surg. 9:353, 1994) tosimple lacerations in the pediatric population (Penoff, Plast. Reconstr.Surg. 103:730, 1999). One study has found that cyanoacrylate tissueadhesive may be the preferred method in terms of cosmetic appearance forthe cutaneous closure of facial lacerations oriented against Langer'slines (Simon et al., J. Emerg. Med. 16:185, 1998).

In a study of the healing of full-thickness wounds in pigs, a dermalmatrix consisting of native bovine collagen coated with elastinhydrolysate was found to serve as a template for dermal tissueregeneration in combination with an epidermal transplantation. Thiscombination treatment reduced wound contraction and improved tissueregeneration (Lamme et al., J. Histochem. Cytochem. 44:1311, 1996).

U.S. Pat. No. 4,837,024, issued Jun. 6, 1989, to Michaeli et al.,discloses an article to promote healing of a surface wound. A suspensionof particles of collagen and a glycosaminoglycan is contacted with thewound surface. Collagen is a major component of the ECM and helps topromote wound healing. The glycosaminoglycan is chemotactic offibroblasts and/or endothelial cells. The collagen/glycosaminoglycan isapplied to the wound and maintained in contact with the wound for anextended period of time, i.e., during the entire healing process oruntil at least closure of the wound by new tissue. The applicationpromotes the vascularization of the wound, attracts fibroblasts andendothelial cells, and generally provides a favorable environment forthe cells during the healing process.

U.S. Pat. No. 5,614,587, issued Mar. 25, 1997, to Rhee et al., and U.S.Pat. No. 5,744,545, issued Apr. 28, 1998, to Rhee et al., disclose acomposition suitable for use as a bioadhesive and a method for usingsuch a composition. The composition comprises fibrillar collagen, afiber disassembly agent, and a multifunctionally activated synthetichydrophilic polymer. The collagen and polymer are mixed to initiatecross-linking, the collagen-polymer mixture is then applied to a firstsurface before substantial cross-linking has occurred, and then a secondsurface is brought in contact with the first surface. The composition isoptically clear so that it could be used in ophthalmic applications, andthe composition comprises biocompatible, non-immunogenic componentswhich leave no toxic, potentially inflammatory or immunogenic reactionproducts at the tissue site of administration.

An invention which would promote healing and lessen scarring would be ofgreat value.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods useful in thepromotion of wound healing. These compositions comprise virgin monomersof tropoelastin and the cross-linking enzyme, lysyl oxidase. The methodcomprises mixing these two components of the composition together andapplying them to a wound before substantial cross-linking has occurred.The tropoelastin monomers and lysyl oxidase only come in contact witheach other immediately before application to the wound or duringapplication to the wound.

Without wishing to be bound by any particular theory, we propose thatthe lysyl oxidase catalyzes the oxidative deamination of the lysineresidues of the tropoelastin monomers at the site of the wound. Then ina non-enzymatic step, cross-links form between the tropoelastin monomersas well as between tropoelastin monomers and other proteins of theextracellular matrix such as collagen. The cross-linked elastin at thesite of injury helps to hold the injured tissue together and therebypromotes healing. The elastin is also chemotactic for fibroblasts,endothelial cells, and inflammatory cells, thereby promoting healing inanother manner. Elastin at the site of injury also helps to lessenscarring since scar tissue is devoid of elastin, and elastin is animportant component of uninjured skin. The cross-linked elastin alsogenerally provides a favorable environment for the cells thatparticipate in the healing process.

In preferred embodiments of the invention, one or both of thetropoelastin monomers and lysyl oxidase are made recombinantly andpurified to homogeneity using standard techniques. The purifiedtropoelastin and lysyl oxidase may then be suspended in a liquid, suchas an aqueous solution (e.g., water or saline) or an organic solvent, orprovided in a dry powder form, or in a lyophilized form. These twocomponents, tropoelastin and lysyl oxidase, are kept separate from eachother until right before use. In another embodiment, the lysyl oxidaseis kept in an inactive form in the presence of the tropoelastin, and thelysyl oxidase not activated until right before use. In yet anotherembodiment, the two components are applied to the wound separately.

In the method of the present invention, the composition may be appliedonly once at the time of the injury or more than once over the course ofwound healing. The composition of tropoelastin and lysyl oxidase mayalso be used in conjunction with sutures, staples, or adhesive strips inclosing the wound.

The composition of the present invention may also be used in promotingthe healing of wounds involving structures with elastic fibers such asarteries, lung tissue, or skin. In particular, the composition may beused in surgeries involving arteries, lungs, or the skin.

In another embodiment of the present invention, the tropoelastincomprises only portions of the tropoelastin protein, preferablycontaining at least one cross-linking domain. In another embodiment, thelysyl oxidase comprises only an active portion of the enzyme.

The preferred compositions of the present invention are biocompatible,non-toxic, and non-immunogenic, and potentially inflammatory orimmunogenic reaction products at the site of administration are avoided.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the reaction catalyzed by lysyl oxidase, the oxidativedeamination of lysine. A variety of non-enzymatic cross-linkingreactions and their products are also shown.

DEFINITIONS

Animal refers to human as well as non-human animals. Non-human animalsinclude mammals, birds, reptiles, amphibians, and fish. Preferably, thenon-human animal is a mammal (i.e., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, or a pig). The term includes transgenic animals.

Biocompatible refers to material that is not toxic to the body, is notcarcinogenic, and does not induce inflammation in body tissues.

Biodegradable refers to material that is degraded by normal bodilyprocesses resulting in products which are readily disposable by the bodyand do not accumulate in the body.

Elastin refers to the cross-linked extracellular matrix protein. Invivo, tropoelastin monomers are cross-linked by lysyl oxidase to formelastin.

Isolated refers to a protein being substantially purified away fromcontaminating matter. In a preferred embodiment, the protein is greaterthan 70% pure, greater than 80% pure, or greater than 90% pure. In aparticularly preferred embodiment, the protein is greater than 95% pureor 98% pure.

Lysyl oxidase refers to a catalyst which is able to catalyze theoxidative deamination of the amino acid lysine as shown in FIG. 1. Inpreferred embodiments of the invention, the catalyst is a lysyl oxidaseprotein enzyme. The genes encoding such enzymes have been cloned from avariety of organisms (Hämäläinen et al., Genomics 11:508, 1991; Trackmanet al., Biochemistry 29:4863, 1990; incorporated herein by reference).In accordance with the present invention, the lysyl oxidase employed ispreferably selected to match as closely as possible the individualsubject to which the compositions are to be applied. For example, humanlysyl oxidase, which has been cloned and characterized (Hämäläinen etal. “Molecular Cloning of Human Lysyl Oxidase and Assignment of the Geneto Chromosome 5q23.3-31.2” Genomics 11(3):508-516, 1991; incorporatedherein by reference) is preferably utilized in the treatment of woundsinvolving human tissue. However, in other embodiments the lysyl oxidasemay be obtained from any species. When compared to the lysyl oxidaseprotein of the target species, useful analogs of lysyl oxidase generallyexhibit at least 60% homology, preferably at least about 70% homology,more preferably at least about 80% homology, and most preferably atleast 90%, 95%, or 99% homology, with a segment of 20 amino acidresidues, preferably with more than 40 amino acid residues, morepreferably yet with substantially the entire sequence of the targetspecies lysyl oxidase. For compositions to be applied to humans, it isparticularly preferred that the lysyl oxidase shows homology to asegment from residues 153-417 and residues 201-417 of the sequence ofhuman lysyl oxidase.

The lysyl oxidase employed in the practice of the present invention maybe modified either chemically or genetically in vivo or in vitro.Examples of non-sequence chemical modifications include phosphorylation,acetylation, methylation, carboxylation, hydroxylation, andglycosylation. Examples of genetic modifications include changes in theamino acid sequence, truncation of the polypeptide chain, addition of atleast one amino acid, and deletion of at least one amino acid.Techniques for producing such chemical and/or genetic modifications arewell known in the art. Any modified protein or polypeptide that retainsthe ability to catalyze the oxidative deamination of lysine ontropoelastin is useful in the practice of the present invention.

Preferred analogs of lysyl oxidase include human lysyl oxidase orbiologically active fragments thereof, whose sequence differ from thewild type by one or more conservative amino acid substitutions or by oneor more non-conservative amino acid substitutions, deletions, orinsertions which do not abolish lysyl oxidase biological activity.Conservative substitutions typically include the substitution of oneamino acid for another with similar characteristics (i.e., substitutionswithin the following groups: valine, glycine; glycine, alanine; valine,isoleucine, leucine; aspartic acid, glutamic acid; asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine). Other conservative substitutions are known by those skilledin the art.

In still other embodiments of the invention, lysyl oxidase may be anon-polypeptide catalyst with the ability to catalyze the oxidativedeamination of lysine.

Protein, peptide, or polypeptide refers to a polymer of amino acids, andthese terms are used interchangeably. The polymer may include natural orunnatural amino acids. The protein or polypeptide may be produced invitro or in vivo via natural, recombinant, synthetic, or other means.The protein or polypeptide may have post-translational modifications ormay have been modified chemically to include phosphorylation,glycosylation, farnesylation, acetylation, methylation, oxidation ofthiols, etc.

Recombinant can refer to organisms, cells, nucleic acids, and proteins.Recombinant cells and organisms are cells and organisms containingrecombinant DNA. Recombinant DNA refers to a nucleic acid sequence whichis not normally found in nature. Usually this term refers to two or morepieces of DNA spliced together to form an unnatural product. Recombinantprotein is protein produced from recombinant DNA (i.e., a nucleic acidwhich differs from that which occurs in nature). In producing arecombinant protein, the regulatory sequences of the gene encoding theprotein are usually different than the ones that occur in the naturalgene. The gene may also have been placed in an organism which normallydoes not possess the gene in order to produce that protein in thedesired organism.

Target species refers to the species on which the composition will beapplied. A target species can be any animal including humans.Preferably, the target species is mammalian, more preferably a domesticmammal (e.g., dog, cat, cow, horse, rabbit, goat, hamster) or a rodent(e.g., rat, mouse), and most preferably human.

Tropoelastin refers to monomer polypeptides which, when cross-linked,form elastin. The genes encoding tropoelastin have been cloned from avariety of organisms (Bressan et al. “Repeating structure of chicktropoelastin revealed by complementary DNA cloning” Biochemistry26:1497-1503, 1987; Raju et al. “Primary structures of bovine elastin a,b, and c deduced from the sequences of cDNA clones” J. Biol. Chem.262:5755-5762, 1987; Indik et al. “Alternative splicing of human elastinmRNA indicated by sequence analysis of cloned genomic and complementaryDNA” Proc. Natl. Acad. Sci. USA 84:5680-5684, 1987; Yeh et al.“Structure of the bovine elastin gene and S1 nuclease analysis ofalternative splicing of elastin mRNA in the bovine nuchal ligament”Biochemistry 28:2365-2370, 1989; Pierce et al. “Heterogeneity of rattropoelastin mRNA revealed by cDNA cloning” Biochemistry 29:9677-9683,1990; each of which is incorporated herein by reference). In accordancewith the present invention, the lysyl oxidase employed is preferablyselected to match as closely as possible the individual subject to whichthe compositions are to be applied. In preferred embodiments of thecurrent invention, the human tropoelastin is utilized in the treatmentof a wound involving human tissue. A variety of different isoforms ofhuman tropoelastin are produced in nature (by alternative splicing); anysuch isoform, or collection of isoforms, may be utilized in the practiceof the present invention. In other embodiments, non-human tropoelastinmay be employed; however, it is generally desirable to match the speciesof animal being treated to the species of the tropoelastin being used.The tropoelastin protein may be modified, as compared with naturallyoccurring human tropoelastin protein, either chemically or geneticallyin vivo or in vitro. When compared to the tropoelastin protein of thetarget species, useful analogs of tropoelastin generally exhibit atleast 60% homology, preferably at least about 70% homology, morepreferably at least about 80% homology, and most preferably at least90%, 95%, or 99% homology, with a segment of 20 amino acid residues,preferably with more than 40 amino acid residues, more preferably yetwith substantially the entire sequence of human tropoelastin. Examplesof non-sequence chemical modifications include phosphorylation,acetylation, methylation, carboxylation, hydroxylation, andglycosylation. Examples of genetic modifications would be changes in theamino acid sequence, truncation of the polypeptide chain, addition of atleast one amino acid, and deletion of at least one amino acid.Techniques for producing such chemical and/or genetic modifications arewell known in the art. Tropoelastin can be prepared recombinantly or bychemical synthesis, but it cannot be purified from natural sourcesbecause it will have already been crosslinked.

Wild type refers to a nucleic acid sequence or amino acid sequence whichis found in nature and has not been mutated. In a preferred embodiment,the wild type sequence is one of the more common sequences for aparticular gene or protein found in a particular species. In someinstances, there may be several wild type sequences for a protein orgene for one particular species due to multiple alleles of a gene ormultiple isoforms of a protein.

Wound refers to damaged biological tissue in the most general sense. Ina preferred embodiment, the wound is a laceration of the skin. In otherembodiments, the wound may be an abrasion of the skin without twoseparated parts of tissue which need to be brought together. In stillother embodiments, the wound may refer to a surgical incision. In otherpreferred embodiments, the wound may involve damage to lung tissue,arterial walls, or other organs with elastic fibers.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As described above, the present invention provides compositions andmethods for promoting wound healing. These compositions comprisetropoelastin and lysyl oxidase. The method of the present inventioninvolves contacting these two components together and applying themixture to a wound before substantial cross-linking of the tropoelastinhas taken place. In another embodiment, the tropoelastin and lysyloxidase are applied separately to the wound. A key aspect of theinvention is that the tropoelastin must be applied to the wound beforesubstantial cross-linking of the tropoelastin has occurred. To thoseskilled in the art, a variety of ways of applying the two substanceswherein the tropoelastin is not substantially cross-linked will beclear. With the cross-linking reaction taking place at the wound site,cross-links will form that will hold the tissue together. Also, the newformation of elastin will attract fibroblasts, inflammatory cells, andendothelial cell by chemotaxis, will result in less scarring, and willprovide a suitable environment for the cells involved in the healingprocess.

Elastin

Elastic fibers in the extracellular space of the lungs, dermis, andlarge blood vessels contribute to the elasticity and resilience of thesetissues. These elastic fibers may comprise only a small (2-4%) portionof the dry weight of skin but may comprise greater than 50% of the dryweight of large arteries (Rosenbloom et al., FASEB J. 7:1208, 1993).These fibers are composed of amorphous elastin and microfibrils.Microfibrils are a complex of glycoproteins organized as small, 10- to12-nm-diameter fibrils and serve as the scaffold onto which elastin isassembled. Elastin is the major component (>90%) of the mature elasticfibers and has been well characterized. The elastin protein is initiallysynthesized as tropoelastin monomers, soluble polypeptides of ca. 72 kDa(Parks et al., Am. J. Respir. Cell Mol. Biol. 2:399, 1990).

The tropoelastin gene exists as a single copy in the genome. The genehas been isolated from several species, including human, bovine, chick,and rat (Bressan et al. “Repeating structure of chick tropoelastinrevealed by complementary DNA cloning” Biochemistry 26:1497-1503, 1987;Raju et al. “Primary structures of bovine elastin a, b, and c deducedfrom the sequences of cDNA clones” J. Biol. Chem. 262:5755-5762, 1987;Indik et al. “Alternative splicing of human elastin mRNA indicated bysequence analysis of cloned genomic and complementary DNA” Proc. Natl.Acad. Sci. USA 84:5680-5684, 1987; Yeh et al. “Structure of the bovineelastin gene and S1 nuclease analysis of alternative splicing of elastinmRNA in the bovine nuchal ligament” Biochemistry 28:2365-2370, 1989;Pierce et al. “Heterogeneity of rat tropoelastin mRNA revealed by cDNAcloning” Biochemistry 29:9677-9683, 1990; each of which is incorporatedherein by reference); the entire bovine and human elastin genes havebeen isolated and sequenced (Indik et al., Proc. Natl. Acad. Sci. USA84:5680, 1987; Yeh et al., Biochemistry 28:2365, 1989; Bashir et al., J.Biol. Chem. 264:8887, 1989; each of which is incorporated herein byreference). Elucidation of the amino acid sequence indicates alternatingsegments of cross-link domains and hydrophobic domains. The cross-linkdomains are characterized by the presence of lysine residues separatedby two to three alanine residues, and the hydrophobic domains are foundto be rich in hydrophobic residues such as glycine, alanine, valine, andproline. This composition results in hydrophobic interactions which arethought to be responsible for the elasticity of the fibers. Thecross-link and hydrophobic domains are encoded by separate exons of thegene.

Extensive alternative splicing of the primary tropoelastin transcriptyields the diversity of tropoelastin isoforms seen at the protein level.The human tropoelastin gene consists of 34 separate exons spanning atotal of ca. 45 kB of genomic DNA. Six of these exons have been reportedto be subject to alternative splicing (Uitto et al., Biochem. Soc.Transact. 19:824, 1991).

Newly synthesized pre-tropoelastin (containing the signal peptide)undergoes intracellular post-translational modifications includinghydroxylation of certain proline residues to form 4-hydroxyproline andremoval of the signal peptide. The tropoelastin monomers are thensecreted into the extracellular milieu where they assemble intofunctional fibers (fibrillogenesis) and are cross-linked to forminsoluble elastin (Uitto et al., Biochem. Soc. Transact. 19:824-829,1991).

The assembly of elastic fibers (fibrillogenesis) takes place at uniquesites close to the cell membrane, generally in infoldings of the cellsurface. Microfibrils are the first component to appear and are found tobe grouped in small bundles near the plasma membrane. Elastin thenappears as amorphous material in discrete loci within eachmicrofibrillar bundle. The microfibrils are thought to align thetropoelastin molecules so that the cross-linking regions are juxtaposed.Lysyl oxidase then oxidizes the terminal amino groups on the side chainsof the lysine residues of the cross-linking regions. The oxidized sidechains then undergo non-enzymatic condensation reactions to form thecross-links. The notion that tropoelastin monomers are secreted from thecell and diffuse onto the surface of growing fibers seems to beinadequate to explain the efficiency of the whole process and thevariable forms of elastic fibers in different tissues. Rather,increasing evidence has supported the idea that helper proteins areneeded in the secretion and fiber assembly steps. Given thattropoelastin monomers produced in organisms are rapidly incorporatedinto insoluble elastin fibers, purification of tropoelastin fromanything other than recombinant sources is not practical. Thus, inaccordance with the present invention, virgin tropoelastin monomers maybe provided by any available method, including, for example, chemicalsynthesis and standard recombinant techniques known in the art.

In one preferred embodiment, the tropoelastin monomers are synthesizedusing available chemical synthetic methods. For example, tropoelastinmonomers can be synthesized using an appropriate solid state syntheticprocedure (Steward et al., Solid Phase Synthesis, Freemantle, SanFrancisco, Calif., 1968). A preferred method is the Merrifield process(Merrifield, Recent Prog. Hormone Res. 23:451, 1967).

Alternatively, tropoelastin monomers may be prepared by recombinanttechniques. Techniques for the overexpression and purification ofrecombinant proteins in a wide variety of cell types are well known inthe art (“Gene Expression Technology,” Methods in Enzymology, vol. 185(D. V. Goeddel, ed.), Academic Press Inc., 1990; incorporated herein byreference). Any such techniques can be employed in accordance with thepresent invention. Since tropoelastin monomers are normally secretedfrom the cell, a preferred way to produce the monomers for the presentinvention would be to introduce the gene with the signal sequence intactand under the control of a strong promoter into a cell and grow therecombinant cells in culture. Preferably, the recombinant cells areeukaryotic (i.e., Saccharomyces cerevisiae, Pichia pastoris), morepreferably the cells are of mammalian origin (i.e., COS cell line, CHOcell line), and most preferably the cells are of a human origin. Incertain preferred embodiments of the invention, the recombinant cellsare derived from human fibroblasts. In another preferred embodiment, therecombinant cells are recombinant bacteria (e.g., E. coli). The desiredtropoelastin monomers would be secreted into the media, and the proteincould be purified to homogeneity from the media. To improve the ease atwhich the protein could be purified, the cells expressing thetropoelastin would be grown in serum-free media, thereby minimizing theamount of protein in the media to start with. Production of thetropoelastin monomers in mammalian cells such as fibroblasts wouldinsure correct folding and post-translational modification of theprotein. This could be important since some of the proline residues intropoelastin are hydroxylated posttranslationally.

Once the recombinant tropoelastin of the present invention is expressed,the protein may be isolated and purified by standard methods includingchromatography (e.g., ion exchange, affinity, size exclusion, andhydroxyapatite chromatography), centrifugation, or differentialsolubility, or by any other available technique for the purification ofproteins (Scopes, Protein Purification Principles and Practice 2ndEdition, Springer-Verlag, New York, 1987; incorporated herein byreference). For immunoaffinity chromatography in particular, the proteinmay be isolated by binding it to an affinity column comprisingantibodies that were raised against that protein and were affixed to astationary support. Alternatively, affinity tags such as an influenzacoat sequence, poly-histidine, or glutathione-S-transferase can beattached to the protein by standard recombinant techniques to allow foreasy purification by passage over the appropriate affinity column.

Any method of producing tropoelastin known in the art can be used aslong as the tropoelastin is able to be cross-linked to form elastin anddoes not initiate negative (e.g., immune reactions) reactions onceintroduced into or applied onto the recipient's body.

If particular isoforms of tropoelastin are needed, the appropriatecDNA(s) can be introduced into the cells for overexpression. Differentisoforms of tropoelastin are found in different individuals within aspecies, in different locations within one individual, and in differenttissues within an individual. In preferred embodiments of the presentinvention, a certain isoform of tropoelastin or mixture of isoforms isused for a particular individual being treated, a particular type ofwound, or a particular type of tissue. For example, it may be desirableto provide a composition comprising multiple different tropoelastinisoforms, whose identity and/or relative quantity are selected to matchas closely as possible the profile of isoforms naturally produced by theindividual to whom the composition is being applied, at the site ofapplication. If the natural isoform distribution in that individual (ora representative thereof, e.g., another member, or an averagedcollection of members, of the same species) and/or at that site, is notknown in advance it may readily be determined by one of ordinary skillin the art using standard techniques such as, for example, genesequencing, restriction fragment length polymorphisms, Western blotting,immunoassay, Northern blotting, Southern Blotting, isoelectric focusing,SDS-polyacrylamide gel electrophoresis, etc. In a preferred embodiment,the individual isoforms of tropoelastin might be prepared separately andmixed at specific ratios later to simulate the ratio of isoforms in aspecific tissue of an individual. The use of tropoelastin isoformsnative to the tissue would minimize any immune reactions.

In another preferred embodiment of the invention, a modified version oftropoelastin is provided which is not susceptible to cross-linking. Thetropoelastin may not be susceptible to cross-linking for a number ofreasons including, for example, its binding another protein or peptide,or its not being folded correctly. In order for the tropoelastin to becross-linked, the binding protein or peptide would need to be removed,or the tropoelastin would need to be refolded. The binding peptide orprotein may be removed by competing off the peptide or protein; cleavageof the binding peptide or protein; conformational change of one of theproteins; etc. If the tropoelastin is not folded correctly, a proteinfrom the family of chaperonins (e.g., GroEL, GroES) or heat shockproteins (e.g., Hsp60, Hsp40, Hsp70) may be added to re-fold thetropoelastin so that it may be cross-linked. In this embodiment, thevirgin tropoelastin monomers and modified lysyl oxidase can be storedtogether without the risk of cross-linking the tropoelastin monomers.

Lysyl Oxidase

The high degree of cross-linking found in the elastic fibers contributesto their proper function. Lysyl oxidase is the enzyme that catalyzes theoxidative deamination of lysine residues leading to the non-enzymaticcondensation of the modified lysine side chains. This same enzyme isinvolved in collagen cross-link formation as well. All but about 5 ofthe 34 lysine residues of tropoelastin participate in some form ofcross-link resulting in a highly insoluble polymer (Rosenbloom et al.,FASEB J. 7:1208, 1993).

Lysyl oxidase is an extracellular, copper-requiring enzyme which hasbeen purified to homogeneity from several animal sources and found tohave a molecular weight of ca. 30 kDa (Stassen, Biochim. Biophys. Acta438:49, 1976; Siegel, Int. Rev. Connect. Tissue Res. 8:73, 1979;Sullivan et al., J. Biol. Chem. 257:13520, 1982; Kagan, Biology ofExtracellular Matrix 1:321, 1986; Kuivaniemi et al., J. Biol. Chem.259:6996, 1984). The genes encoding lysyl oxidase have been cloned fromhuman and rat cDNA libraries (Hämäläinen et al., Genomics 11:508 (1991);Trackman et al., Biochemistry 29:4863 (1990)).

Oxidation of lysine residues in tropoelastin leads to the formation ofα-aminoadipic-δ-semialdehyde (AAS). Reaction of a lysine residue withAAS leads to the formation of a dehydrolysinonorleucine cross-link.Reaction of two AAS residues leads to an Aldol condensation product.Tetrafunctional cross-linkages can be formed in elastin from threepeptidyl aldehydes (AAS) and one unmodified lysine residue (FIG. 1)(Kagan, Path. Res. Pract. 190:910, 1994; incorporated herein byreference).

For the purposes of the present invention, lysyl oxidase can be producedby any available method, including, for example, chemical synthesis,recombinant methods, and purification from a natural source. In apreferred embodiment, lysyl oxidase is produced recombinantly in muchthe same manner as the tropoelastin (discussed supra). Again, lysyloxidase is normally secreted so a preferred method of producing andpurifying the enzyme would be to secrete it into the media and thenpurify the protein from the media. If a modified version (i.e., changein amino acid sequence) was needed, the appropriate cDNA could beintroduced into the cell to produce the desired mutant protein. Standardtechniques for these procedures are known in the art.

In a preferred embodiment of the invention, a modified version of thelysyl oxidase is produced which is inactive initially and cansubsequently be converted into an active form. This particularembodiment of the invention allows the tropoelastin and inactive lysyloxidase to be stored together. Then, prior to application to a wound,the inactive lysyl oxidase is converted to the active form by cleavageof the protein or by a change in pH, temperature, salt concentration,metal ion concentration, etc. The conversion from the inactive form tothe active form might even take place at the site of the wound and becaused by a protease or change in pH at the wound site per se.

In another preferred embodiment of the invention, a modified version oflysyl oxidase would be provided which binds to another protein orpeptide (binding peptide or protein). The binding of this bindingprotein or peptide to the modified lysyl oxidase would lead toinactivation of the lysyl oxidase enzyme. In order for the lysyl oxidaseto regain its enzymatic activity, the binding protein or peptide wouldneed to be removed. Examples of this approach include competing off themodified lysyl oxidase using another peptide or protein, a small organicmolecule, a metal, nucleic acid, polysaccharide, etc.; cleavage of thebinding peptide or protein; conformational change of modified lysyloxidase; and conformational change of the binding protein or peptide.

In a particularly preferred embodiment, a protein from the family ofchaperonins (e.g., GroEL, GroES) or heat shock proteins (e.g., Hsp60,Hsp40, Hsp70) may be used as the binding protein, and the lysyl oxidasemay be modified by techniques well known in the art to include a bindingdomain specific for a chaperonin or heat shock protein (Ranson et al,“Chaperonins” Biochem. J. 333 (Pt. 2):233-242, Jul. 15, 1998; Fink etal., “Chaperone-mediated protein folding” Physiol. Rev. 79(2):425-449,April 1999; each of which is incorporated herein by reference). In thisway, the virgin tropoelastin monomers and modified lysyl oxidase boundto a chaperonin or heat shock protein can be stored together without therisk of cross-linking the tropoelastin monomers. After the tropoelastinand lysyl oxidase are applied to a wound or immediately before they areapplied to a wound, an agent such as a small molecule (e.g., estrogen)is added to restore lysyl oxidase activity and begin the cross-linkingof tropoelastin (Bohen et al., “Hold 'em and fold 'em: chaperones andsignal transduction” Science 268(5125):1303-1304, Jun. 2, 1995;incorporated herein by reference).

Packaging

In one preferred embodiment of the invention, the tropoelastin monomersare be provided in one vial, and the lysyl oxidase are provided inanother vial. These proteins might be provided in a dry powder form, insolid form (i.e., lyophilized), in solution, or in suspension. To theproteins may have been added emulsifiers, salts, preservatives, otherproteins, nucleic acids, protease inhibitors, antibiotics, perfumes,polysaccharides, adhesive agents, polymers, microfibrils, oils, etc.

In another preferred embodiment, the tropoelastin or the lysyl oxidase,or both, is encapsulated in a biodegradable polymer so that when thecomposition is applied to a wound, the polymer degrades and thetropoelastin and/or lysyl oxidase is released; the released tropoelastincan then be crosslinked.

Biodegradable polymers are usually based on functional groups such asesters, anhydrides, orthoesters, and amides. Rapidly biodegradablepolymers include poly[lactide-co-glycolide], polyanhydrides, andpolyorthoesters. Preferred bioerodible polymers include polylactides,polyglycolides, and copolymers thereof, poly(ethylene terephthalate),poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone),poly(lactide-co-glycolide), polyanhydrides, polyphosphazenes,poly(ε-caprolactone), poly(dioxanone), poly(hydroxybutyrate),poly(hydroxyvalerate), polyorthoesters, blends, and copolymers thereof.Examples of biodegradable and biocompatible polymers of acrylic andmethacrylic acids or esters include poly(methyl methacrylate),poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutylmethacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate),poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methylacrylate), poly(isopropyl acrylate), poly(isobutyl acrylate),poly(octadecyl acrylate), etc. Other polymers which can be used in thepresent invention include polyalkylenes such as polyethylene andpolypropylene; polyarylalkylenes such as polystyrene; poly(alkyleneglycols) such as poly(ethylene glycol); poly(alkylene oxides) such aspoly(ethylene oxide); and poly(alkylene terephthalates) such aspoly(ethylene terephthalate). Additionally, polyvinyl polymers can beused which include polyvinyl alcohols, polyvinyl ethers, polyvinylesters, and polyvinyl halides. Exemplary polyvinyl polymers includepoly(vinyl acetate), polyvinyl phenol, and polyvinylpyrrolidone.Mixtures of two or more of the above polymers could also be used in thepresent invention.

Some polymeric materials are known to release entrapped compounds uponexposure to a stimulus such as a change in pH or temperature. Anexamples of microparticles that release as a function of a change in pHinclude the diketopiperazine particles describes in U.S. Pat. No.5,352,461 issued on Oct. 4, 1994, to Steiner et al., and the proteinoidformulations described in U.S. Pat. Reissue No. 35,862, issued on Jul.28, 1998.

Application

Methods for using the composition include, for example, mixing the twoseparate components together immediately before application to the wound(i.e., before substantial cross-linking has occurred). Other preferredembodiments include applying either the tropoelastin or lysyl oxidaseand then the other one to the wound site. One particularly preferredembodiment involves application of a composition comprising lysyloxidase and tropoelastin to a wound and then use of sutures, staples,adhesive strips, or tissue adhesive to hold the tissue together duringthe healing process.

Another particularly preferred embodiment involves application of thecomposition more than once during the healing process. Preferredregiments for applying the lysyl oxidase and tropoelastin includeseveral times a day, once a day, once a week, twice a week, once amonth, and twice a month. In following each of these regiments, theagents would be mixed immediately before application, or they would beapplied separately to the wound.

The lysyl oxidase and tropoelastin are delivered to the wound site usingany means to apply a liquid, paste, gel, or solid (e.g., powder). Thesemeans include, for example, a brush, a syringe, a spatula, and acontainer specifically designed to delivery the agents such as a tubewith a narrow tip.

In another preferred embodiment of the invention, the composition isapplied to a wound resulting from deep trauma or surgery involving thelungs, large arteries, or other tissues with significant amounts ofelastic fibers.

OTHER EMBODIMENTS

The foregoing has been a description of certain non-limiting preferredembodiments of the invention. Those of ordinary skill in the art willappreciate that various changes and modifications to this descriptionmay be made without departing from the spirit or scope of the presentinvention, as defined in the following claims.

1. A method for promoting wound healing comprising the steps of:providing isolate tropoelastin and isolated lysyl oxidase separated fromeach other; and applying both said tropoelastin and said lysyl oxidaseto wound simultaneously or sequentially.
 2. The method of claim 1wherein tropoelastin is wild type tropoelastin matched to species ofrecipient.
 3. The method of claim 1 wherein tropoelastin is modifiedtropoelastin.
 4. The method of claim 3 wherein amino acid sequence oftropoelastin has been changed relative to amino acid sequence of wildtype tropoelastin.
 5. The method of claim 1 wherein tropoelastincomprises a heterogeneous mixture of tropoelastin isoforms.
 6. Themethod of claim 1 wherein lysyl oxidase comprises an enzymaticallyactive portion of lysyl oxidase.
 7. The method of claim 1 wherein lysyloxidase is modified lysyl oxidase relative to wild type lysyl oxidase.8. The method of claim 7 wherein the amino acid sequence of lysyloxidase has been changed relative to wild type lysyl oxidase.
 9. Themethod of claim 1 wherein the method comprises the additional step of:repeatedly applying the tropoelastin and lysyl oxidase to the woundduring the healing process.
 10. The method of claim 1 wherein the methodcomprises the additional step of: approximating separated tissue of thewound using sutures, staples, adhesive strips, or tissue glue.
 11. Themethod of claim 1 wherein the step of applying comprises applying thetropoelastin and lysyl oxidase with a sterile syringe.
 12. The method ofclaims 1 wherein the tropoelastin or lysyl oxidase has been mixed withother materials selected from the group consisting of polymers,emulsifiers, oils, perfumes, proteins, polysaccharides, nucleic acids,microfibrils, antimicrobial agents, adhesive agents, and proteaseinhibitors.
 13. A kit comprising tropoelastin and lysyl oxidase inseparate compartments.
 14. The kit of claim 13 wherein the tropoelastinis wild type tropoelastin.
 15. The kit of claim 13 wherein thetropoelastin is modified tropoelastin.
 16. The kit of claim 15 whereinthe amino acid sequence of tropoelastin has been changed relative towild type sequence.
 17. The kit of claim 13 wherein the lysyl oxidase iswild type lysyl oxidase.
 18. The kit of claim 13 wherein the lysyloxidase is modified lysyl oxidase.
 19. The kit of claim 18 wherein theamino acid sequence of lysyl oxidase has been changed relative to wildtype sequence.
 20. A kit comprising tropoelastin and lysyl oxidase inthe same compartment, wherein the lysyl oxidase is in an inactive formwhich can be later converted to an active form.
 21. A kit comprisingtropoelastin and lysyl oxidase in the same compartment, wherein eitherthe tropoelastin or lysyl oxidase is encapsulated by a polymer.
 22. Thekit of claim 21 wherein the polymer is biodegradable.
 23. The method ofclaim 1 wherein the wound involves the skin.
 24. The method of claim 1wherein the wound involves an artery.
 25. The method of claim 1 whereinthe wound involves lung tissue.